Hematopoietic stem cell transplantation (HCT) is a life-saving therapy for a variety of hematological disorders. When using allogeneic HCT as a therapy for hematological malignancies, however, the graft- versus-tumor (GVT) effect is in large part associated with graft-versus-host disease (GVHD). Therefore, controlling alloreactivity to prevent GVHD while preserving the GVT effect represents a true dilemma and the key challenge in HCT. The long-term goal of this project is to exploit the ability of foxp3-induced regulatory T cells (Tregs) to contain alloreactive T cells and to prevent GVHD while retaining the GVT effect. The central hypothesis is that forced foxp3 expression will convert a T cell clone into Tregs with a known TCR specificity, which can be activated by a specific antigen. Because the onset and duration of activation can be regulated in antigen-specific T cells, the suppressive function of these Tregs can be controlled at will. Since published data by others indicate that the GVT effect can be spared while GVHD is suppressed by Tregs, this strategy is expected to have the potential to control GVHD while preserving the GVT effect. Three specific aims are designed to test the central hypothesis: 1) Prevent GVHD by foxp3-induced Tregs that express transgenic (Tg) TCR. CD4+CD25- cells will be isolated from WT or TCR Tg mice and converted into Tregs by foxp3-transduction via retroviral infection. The effects of WT or TCR Tg Tregs will be tested in the prevention of lethal GVHD after activation with recipient alloantigen. 2) Control GVHD by foxp3-induced Tregs that are antigen-specific cloned T cells. An antigen-specific T-cell clone will be transduced with foxp3 and used as Tregs. The effects of foxp3-expressing T cell clone will be tested in the prevention and treatment of GVHD after activation by recipient alloantigen or a peptide antigen. 3) Distinguish GVHD and the GVL effect by antigen-specific, foxp3-induced Tregs. In GVHD plus tumor models, the ability of Tregs to control GVHD while maintaining GVT effect will be compared under two conditions where Tregs are constantly activated by recipient alloantigen or transiently activated by a peptide antigen. The goal of this proposed research is to permit donor immune cells fighting against tumor, while limiting their ability to cause injury on normal tissues in the patient after bone marrow transplantation (BMT). If successful, the research would substantially enhance the therapeutic potential of BMT for the treatment of leukemia and other blood-borne diseases. [unreadable] [unreadable]